Cardiovascular safety assay

ABSTRACT

The present invention provides assays and kits for the screening of test compounds for their capability to induce cardiotoxicity in a subject. Said assays and kits are based on the finding that the interaction of astemizole with the HERG potassium channel can be exploited to predict cardiotoxicity of compounds during the development of new therapeutics and other agents.

The present invention relates to the field of cardiovascular safety assays and provides assays and kits for the screening of test compounds for their capability to induce cardiotoxicity in a subject. Said assays and kits are based on the finding that the interaction of astemizole with the HERG potassium channel can be exploited to predict potential cardiotoxicity of compounds during the development of new therapeutics and other agents. The present invention finds particularly advantageous use in high throughput screening of chemical compound libraries.

BACKGROUND OF THE INVENTION

Evidence has accrued that several drugs may prolong cardiac repolarisation (hence, “measured as” the QT interval of the surface electrocardiogram) to such a degree that potentially life-threatening ventricular arrhythmias e.g. torsades de pointes (TdP) may occur, especially in case of overdosage or pharmacokinetic interaction.

The number of drugs reported to induce QT interval prolongation with or without TdP continues to increase (W. Haverkamp et al. (2000) Cardiovascular Research 47, 219-233). As many as 50 clinically available or still investigational non-cardiovascular drugs and cardiovascular non-anti-arrhythmic drugs have been implicated. A number of drugs, both old and new, have either been withdrawn from the market or have had their sale restricted. Of concern is the interval, usually measured in years, from the marketing of these drugs to initial recognition of their association with QT interval prolongation and/or TdP. It would therefore be beneficial to investigate any new chemical entity for this potential side effect before its first use in man at an early stage of the development of new therapeutics and/or other agents.

A key component in the present development of new therapeutic agents consists of High Throughput Screening (HTS) of chemical compound libraries. Pharmaceutical companies have established large collections of structurally distinct compounds, which act as the starting point for drug target lead identification programs. A typical corporate compound collection now comprises between 100,000 and 1,000,000 discrete chemical entities. While a few years ago a throughput of a few thousand compounds a day and per assay was considered to be sufficient, pharmaceutical companies nowadays aim at ultra high throughput screening techniques with several hundreds of thousands of compounds tested per week. In a typical HTS related screen format, assays are performed in-multi-well microplates, such as 96,384 or 1536 well plates. The use of these plates facilitates automation such as the use of automated reagent dispensers and robotic pipetting instrumentation. Further, to reduce the cycle time, the costs and the resources for biochemicals such as recombinant proteins, HTS related screens are preferably performed at room temperature with a single measurement for each of the compounds tested in the assay.

A decisive criterion in the lead evaluation process will be an early recognition of their potential association with QT prolongation and/or TdP. However, there are currently no reliable, fast, easy screening methods available to assess cardiotoxicity, which can cope with the number of compounds identified in the currently deployed HTS techniques. It is an object of this invention to solve this problem in the art by providing assays and kits which are based on the finding that the interaction of astemizole with the HERG potassium channel can be exploited to predict cardiotoxicity of compounds during the development of new therapeutics and other agents.

The currently available in vitro models comprise heterologous expression systems, disaggregated cells, isolated tissues and the isolated intact (Langendorf-perfused) heart. In all models the effect of potassium current blockade is assessed by measurement of either ionic currents using two-electrode voltage clamp recordings (Dascal N. (1987) Crit. Rev. Biochem 22, 341-356) or patch-clamp recordings (Zhou Z. et al., (1998) Biophysical Journal 74, 230-241), of membrane potentials using microelectrodes or confocal microscopy (Dall'Asta V. et al. (1997) Exp. Cell Research 231, 260-268). None of the aforementioned methods can be used in an HTS screen in view of the complexity of the experimental procedures, the slow cycling times, the nature of the source materials (i.e. isolated tissues and disaggregated cells thereof) and the reliability of the test results.

The present inventors surprisingly found that a binding assay using labeled astemizole as a specific ligand for the HERG channel can be used to predict the potential association of compounds with QT prolongation and/or TdP. This binding assay solves the aforementioned problems and can be deployed in an HTS related screen format.

A similar assay has been described by Chadwick C. et al. (Chadwick C. et al., (1993) Circulation Research 72, 707-714) wherein [³H]-dofetilide has been identified as a specific radioligand for the cardiac delayed rectifier K⁺-channel. This article further demonstrates a good correlation between dofetilide displacement and potassium channel blocking activity of a number of antiarrhytmic compounds. This binding assay facilitates the characterization of drug-channel interactions at the molecular level.

In this assay labeled dofetilide has been prepared from the dibromo precursor by ³H-exchange yielding the incorporation of two ³H-labels per molecule. There is a direct correlation between the number of ³H-labels per molecule and the sensitivity of the binding assay. The present invention provides an improved binding assay over the above, as the use of a desmethylastemizole precursor in a reaction with [3³H]-methyliodide resulted in the incorporation of three ³H-labels per molecule astemizole. The specific activity of the thus obtained radioligand is 1.5-2 times higher than the specific activity of [³H]-dofetilide.

Further, the dofetilide assay could not be used in an HTS related screen format since the ventricular myocytes isolated from adult male guinea pigs had to be used within 6 hours of isolation. In addition only 36% of the isolated cells were viable and could be used in the binding assay. In order to be used in an HTS related screen, the starting material should be readily available and in sufficient amounts. The present invention solves this problem as membrane preparations of HEK293 cells, stably expressing the HERG potassium channel, are used. Said cells can be maintained in culture in sufficient amount avoiding the need and supply of animal models and as cell membranes are used in the binding assay, the latter can be stored in binding assay ready aliquots at −80° C. for later use. A further drawback of the dofetilide binding assay described by Chadwick et al. has to do with the incubation protocol. As viable myocytes are used, the incubation has to be performed at the physiological temperature of 34° C. The latter undoubtedly increases the costs, possible cycle time and complexity of the assay if to be performed in an HTS related screen format. The present invention solves this problem as it was surprisingly demonstrated that the membrane preparations could be incubated at room temperature. Espescially in light of a study by Zhoe Z. et al. Zhou Z. et al., (1998) Biophysical Journal 74, 230-241) which concluded that the kinetic properties measured for HERG are markedly dependent on temperatura and that differences observed in several reports, are diminished when studies are performed at physiological temperatures, i.e. 35° C.

This and other aspects of the invention will be described herein below.

SUMMARY OF THE INVENTION

The present invention provides an assay for screening test compounds for their capability to induce cardiotoxicity in a subject, the method comprising incubating a source containing HERG or a fragment thereof with a reference compound and the test compound, for a time sufficient to allow binding of the reference compound and of the test compound with the HERG polypeptide channel and measuring the effect of the test compound on the amount of reference compound bound to HERG.

In a preferred embodiment of this invention, the assay consists of incubating membrane preparations of cells, preferably EK293 cells, expressing on the surface thereof the HERG polypeptide channel comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof; with a labeled reference compound. Wherein said labeled reference compound is a drug capable to induce cardiac arrhythmia in a subject, preferably said labeled reference compound is [³H]-astemizole. Incubating the above together with the test compound and measure the effect of the test compound on the amount of reference compound bound to the HERG polypeptide channel. In a further embodiment the means of measurement consist of separating means to remove the excess of unbound labeled reference compound from the incubation mixture and of means for detection of the labeled reference compound wherein the latter preferably consists of radiolabeled measurement using scintillation counting.

The invention further provides a high-throughput assay for screening compounds for their capability to induce cardiotoxicity in a subject, the assay comprising;

-   -   a) contacting membrane preparations of cells expressing on the         surface thereof HERG polypeptide channels having an amino acid         sequence that is at least 80% identical to that of SEQ ID NO:2         or fragments thereof, with a labeled reference compound for a         time sufficient to allow binding of the reference compound with         the HERG polypeptide channel;     -   b) contacting membrane preparations of cells expressing on the         surface thereof HERG polypeptide channels having an amino acid         sequence that is at least 80% identical to that of SEQ ID NO:2         or fragments thereof, with the labeled reference compound of         step a) together with the test compound for a time sufficient to         allow binding of the reference compound and of the test compound         with the HERG polypeptide channel;     -   c) measuring the amount of labeled reference compound bound to         the HERG channel in step a);     -   d) measuring the amount of labeled reference compound bound to         the HERG channel in step b); and     -   e) compare the amount of labeled reference compound bound to the         HERG channel measured in step a) with the amount of labeled         reference compound bound to the HERG polypeptide channel         measured in step b).         In a preferred embodiment of the high-throughput screening         assay, the membrane preparations are derived from cells,         preferably HEK293 cells, expressing on the surface thereof HERG         polypeptide channels encoded by the amino acid sequence         consisting of SEQ ID NO:2. In a further embodiment of the         high-throughput screening assay the labeled reference compound         is astemizole, preferably [³H]-astemizole.

The present invention also encompasses kits for screening compounds for their capability to induce cardiotoxicity in a subject as well as the use of reagents, including polynucleotides, polypeptides and suitable reference compounds in the assays of the present invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 A shows the saturation binding of [³H]-astemizole to cell membrane preparations of HEK293 cells stably transfected with the HERG channel cDNA. TB represents Total Binding measured, NSB represents Non Specific Binding measured and SB represents Specif Binding measured.

FIG. 1B shows the Scatchard plot for the saturation binding experiments. From the fitted line a K_(D) of 3.07±2.26 nM (n=11) could be determined with a B_(max) (Maximal Binding) of 3260±900 fmol/mg protein (n=11).

FIG. 2 shows the binding affinities of 42 reference compounds compared to the electrophysiological patch clamp data. A Spearman rank correlation coefficient of 0.87 could be obtained.

DETAILED DESCRIPTION

The present invention relates to the field of cardiovascular safety assays and provides assays and kits for the screening of test compounds for their capability to induce cardiotoxicity in a subject. Said assays and kits are based on the finding that the interaction of astemizole with the HERG potassium channel can be exploited to predict cardiotoxicity of compounds during the development of new therapeutics and other agents. The present invention finds particularly advantageous use in high throughput screening of chemical compound libraries.

In one embodiment of the present invention, the assay for screening test compounds comprises: a) incubating a source containing HERG or a fragment thereof with i) a reference compound, ii) the test compound; and b) measuring the effect of the test compound on the amount of reference compound bound to HERG.

In a specific embodiment of the present invention the assays are used to assess the capability of the test compounds to induce cardiac arrhythmia in a subject.

As used herein the term “test compound” refers to a chemically defined molecule whose cardiac arrhythmia inducing capability is assessed in an assay according to the invention. Test compounds include, but are not limited to, drugs, ligands (natural or synthetic), polypeptides, peptides, peptide mimics, polysaccharides, saccharides, glycoproteins, nucleic acids, polynucleotides and small organic molecules. In one embodiment test compounds comprise an existing library of compounds. In another embodiment, test compounds comprise a novel library of compounds.

As used herein the term “reference compound” refers to a drug capable to induce cardiotoxicity in a subject. Reference compounds include, but are not limited to, astemizole, terfenadine, erythromycin, sparfloxain, probucol, terodiline and sertindole.

As used herein the term “HERG” refers to the Human Ether-a-go-go-Related Gene channel. It is a delayed rectifier potassium channel that plays a role in the control of action potential repolarization in many cell types. HERG was originally cloned from human hippocampus and it is strongly expressed in the heart. The HERG polypeptides according to the invention include isolated and purified proteins having an amino acid sequence that is at least 80% identical to that of SEQ ID NO:2 or a fragment thereof. In a further embodiment the HERG polypeptide channel according to the invention has an amino acid sequence comprising the amino acid sequence of SEQ ID NO:2. In a preferred embodiment the HERG polypeptide according to the invention consists of SEQ ID NO:2.

Variants of the defined sequence and fragments also form part of the invention. Variants include those that vary from the parent sequence by conservative amino acid changes, wherein “conservative amino acid changes” refers to the replacement of one or more amino acid residue(s) in the parent sequence without affecting the biological activity of the parent molecule based on the art recognized substitutability of certain amino acids (See e.g. M. Dayhoff, In Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3, pgs 345-352, 1987). Further variants are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted or added in any combination.

Methods for comparing the identity and similarity of two or more sequences are well known in the art. Thus for instance, programs available in the Winconsin Sequence Analysis Package, version 9.1 (Devreux J. et al, Nucleic Acid Res., 12, 387-395, 1984), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % similarity between two polypeptide sequences. BESTFIT uses the “local homology” algorithm of Smith and Waterman (J. Mol. Biol., 147, 195-197, 1981) and finds the best single region of similarity between two sequences. BESTFIT is more suited to compare two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer. In comparison, GAP aligns two sequences, finding a “maximum similarity”, according to the algorithm of Neddleman and Wunsch (J. Mol. Biol., 48, 443-453, 1970). GAP is more suited to compare sequences that are approximately the same length and an alignment is expected over the entire length. Preferably, the parameters “Gap Weight” and “Length Weight” used in each program are 50 and 3 for polynucleotide sequences, and 12 and 4 for polypeptide sequences, respectively. Preferably, % identities and similarities are determined when the two sequences being compared are optimally aligned. Other programs for determining identity and/or similarity between sequences are also known in the art, for instance the BLAST family of programs (Altschul S F et al, Nucleic Acids Res., 25:3389-3402, 1997).

Those skilled in the art will recognize that the polypeptides according to the invention, i.e. the HERG polypeptide channel, could be obtained by a plurality of recombinant DNA techniques including, for example, hybridization, polymerase chain reaction (PCR) amplification, or de novo DNA synthesis (See e.g., T. Maniatis et al. Molecular Cloning: A Laboratory Manual, 2d Ed. Chap. 14 (1989)). Thus, in a further embodiment the present invention provides the use of the isolated and purified polynucleotides encoding the HERG polypeptide or a fragment thereof, in an assay or kit according to the invention. In another embodiment the present invention provides the use of the isolated and purified polynucleotide encoding the HERG polypeptide channel or a fragment thereof comprising the polynucleotide sequence of SEQ ID NO:1. In a preferred embodiment the present invention provides the use of the isolated and purified polynucleotide encoding the HERG polypeptide channel consisting of the polynuceotide sequence of SEQ ID NO:1.

The term “fragments thereof” describes a piece, or sub-region of protein or polynucleotide molecule whose sequence is disclosed herein, such that said fragment comprises 5 or more amino acids that are contiguous in the parent protein, or such that said fragment comprises 15 or more nucleic acids that are contiguous in the parent polynucleotide. The term “fragments thereof” is intended to include “functional fragments” wherein the isolated fragment, piece or sub-region comprises a functionally distinct region such as an active site, a binding site or a phosphorylation site of the receptor protein. Functional fragments may be produced by cloning technology, or as the natural products of alternative splicing techniques.

As used herein, “isolated” refers to the fact that the polynucleotides, proteins and polypeptides, or respective fragments thereof in question, have been removed from its in vivo environment so that it can be manipulated by the skilled artisan, such as but not limited to sequencing, restriction digestion, site-directed mutagenesis, and subcloning into expression vectors for a nucleic acid fragment as well as obtaining the protein or protein fragments in quantities that afford the opportunity to generate polyclonal antibodies, monoclonal antibodies, amino acid sequencing, and peptide digestion. Therefore, the nucleic acids as described herein can be present in whole cells or in cell lysates or in a partially, substantially or wholly purified form.

A polypeptide is considered “purified” when it is purified away from environmental contaminants. Thus a polypeptide isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized polypeptide sequence is considered to be substantially purified when purified from its chemical precursors. A “substantially pure” protein or nucleic acid will typically comprise at least 85% of a sample with greater percentages being preferred. One method for determining the purity of a protein or nucleic acid molecule, is by electrophoresing a preparation in a matrix such as polyacrylamide or agarose. Purity is evidenced by the appearance of a single band after staining. Other methods for assessing purity include chromatography and analytical centrifugation.

The term “time sufficient to allow binding” as used herein refers to the time needed to generate a detectable amount of labeled reference compound bound to the HERG polypeptide channel. The time needed to generate this detectable amount will vary depending on the assay system. One of skill in the art will know the amount of time sufficient to generate a detectable amount of labeled reference compound bound to the HERG polypeptide channel based upon the assay system.

Assays

Assays of the present invention can be designed in many formats generally known in the art of screening compounds for binding polypeptide channels.

The assays of the present invention advantageously exploit the fact that the interaction of astemizole with the HERG potassium channel can be exploited to predict cardiotoxicity of compounds during the development of new therapeutics and other agents.

Therefore, the present invention provides an assay for screening test compounds, the assay comprising a) incubating a source containing HERG or a fragment thereof with; i) a reference compound, ii) the test compound; and b) measuring the effect of the test compound on the amount of reference compound bound to HERG.

In a first embodiment of this invention the source containing HERG is an isolated and purified protein which encodes HERG having an amino acid sequence that is at least 80% identical to that of SEQ ID NO:2 or a fragment thereof.

In a second embodiment of this invention the source containing HERG is an isolated and purified protein which encodes HERG comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof.

In a further embodiment of this invention the source containing HERG are cells expressing on the surface thereof the HERG polypeptide channel or a fragment thereof.

In another embodiment of this invention the source containing HERG are membrane preparations of cells expressing on the surface thereof the HERG polypeptide channel or a fragment thereof.

In an alternative embodiment of this invention, the reference compound is a compound capable to induce cardiotoxicity in a subject, preferably selected from the group consisting of astemizole, terfenadine, erythromycin, sparfloxain, probucol, terodiline and sertindole. In a preferred embodiment the reference compound is astemizole. It is a further object of this invention to provide assays wherein the reference compound is labeled, preferably radiolabeled.

In a preferred embodiment, the assay for screening test compounds for their capability to induce cardiotoxicity in a subject consists of a) incubating membrane preparations of cells expressing on the surface thereof HERG polypeptide channels encoded by the amino acid sequence consisting of SEQ ID NO:2 with i) [³H]-astemizole, ii) the compound to be tested; and measuring the effect of the test compound on the amount of reference compound bound to HERG. The label of the reference compound is used to measure this effect wherein said label can be measured using amongst others scintillation counting.

A specific embodiment of the assays according to the invention, consists of an high-throughput assay for screening test compounds, the assay comprising: a) contacting membrane preparations of cells expressing on the surface thereof HERG polypeptide channels having an amino acid sequence that is at least 80% identical to that of SEQ ID NO:2 or fragments thereof, with a labeled reference compound for a time sufficient to allow binding of the reference compound with the HERG polypeptide channel; b) contacting membrane preparations of cells expressing on the surface thereof HERG polypeptide channels having an amino acid sequence that is at least 80% identical to that of SEQ ID NO:2 or fragments thereof, with the labeled reference compound of step a) together with the test compound for a time sufficient to allow binding of the reference compound and of the test compound with the HERG polypeptide channel; c) measuring the amount of labeled reference compound bound to the HERG channel in step a); d) measuring the amount of labeled reference compound bound to theHERG channel in step b); and e) compare the amount of labeled reference compound bound to the HERG channel measured in step a) with the amount of labeled reference compound bound to the HERG polypeptide channel measured in step b).

In a further embodiment the membrane preparations of the high-throughput screening assay consist of membrane preparations of cells expressing on the surface thereof the HERG polypeptide channel comprising the amino acid sequence of SEQ ID NO:2 or fragments thereof.

In a preferred embodiment of this invention the membrane preparations of the high-throughput screening assay consist of membrane preparations of cells, preferably HEK 293 cells, expressing on the surface thereof HERG polypeptide channels consisting of the amino acid sequence of SEQ ID NO:2.

In a further preferred embodiment, the labeled reference compound in the high-throughput screening assay consists of [³H]-labeled astemizole. Said label can be measured using amongst others scintillation counting.

In another specific embodiment the present invention provides a high-throughput proximity detection assay for screening test compounds the assay comprising:

-   -   i) HERG labeled with a first label capable of participating in a         proximity detection assay;     -   ii) a reference compound labeled with a second label capable of         participating in a proximity detection assay;     -   iii) contacting HERG of step i) and a reference compound of         step ii) together with a test compound for a time sufficient to         allow binding of the reference compound and of the test compound         to HERG; and     -   iv) detect an interaction between HERG of step i) and a         reference compound of step ii) by means of proximity of the         first label with the second label when HERG and the reference         compound interact.         The proximity of the first label to the second label, brought         about by the interaction of HERG and the reference compound         results in the production of a detectable signal. This may be         achieved by e.g. a scintillation proximity assay (SPA) system,         in which one of the labels is a radiolabel suitable for use in         SPA and the other label is a fluorescer comprised in a solid         phase. The detectable signal is light energy emitted when the         labeled HERG protein interacts with the labeled reference         compound, bringing the radiolabel sufficiently close to the         fluorescer. Scintillation proximity assay technology is         described in U.S. Pat No. 4,568,649.

Alternatively, the detectable signal may be a change in an existing signal output, eg. fluorescence. Fluorescence resonance energy transfer (FRET) is a method which works on this principle and is described by Tsien R. et al. (Tsien R. et al. (1993) Trends Cell Biol. 3: 242-245). It employs two different fluorescent molecules, a donor and an acceptor, such that when.these are in sufficient proximity to one another the fluorescence of the donor molecule is absorbed by the acceptor molecule and light of another wavelength is emitted. Thus, when there is an interaction between two molecules such as HERG and a reference compound, each of which is labeled with one of these fluorescent molecules, a detectable signal is produced.

By such proximity assays as are described above, the screening assay according to the invention may be performed in a single step, i.e. without the need of a separation step to remove the excess of labeled reference compound from the incubation mixture using separation means such as filtration.

In a preferred embodiment of the high-throughput proximity detection assay, HERG is labeled with the fluorescer comprised in a solid phase, such as coated scintillation proximity assay beads and the reference compound is labeled with the radiolabel preferably the reference compound is radiolabeled astemizole of formula (III).

It will be readily appreciated by the skilled artisan that the binding of astemizole with HERG may also be used in a method for the structure-based or rational design of compound which affects the aforementioned binding, by:

-   -   a) probing the structure of the binding site of the HERG         polypeptide channel with astemizole;     -   b) identifying contacting atoms in the binding site of the HERG         polypeptide channel that interact with astemizole during         binding;     -   c) design test compounds that interact with the atoms identified         in (b) to affect the HERG—astemizole interaction; and     -   d) contact said designed test compound with a source containing         HERG or a fragment thereof, to measure the capability of said         compound to affect the amount of labeled astemizole bound to         HERG.         It will be further appreciated that this will normally be an         iterative process.         Kits

The present invention also provides kits that can be used in the above assays. In one embodiment the kit comprises a) a source containing HERG; b) a reference compound.

In a first embodiment the kit comprises a source containing HERG selected from: i) an isolated and purified protein which encodes HERG having an amino acid sequence that is at least 80% identical to that of SEQ ID NO:2 or a fragment thereof; ii) an isolated and purified protein which encodes HERG comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof; iii) cells expressing on the surface thereof the HERG polypeptide channel or a fragment thereof; or iv) membrane preparations of cells expressing on the surface thereof the HERG polypeptide channel or a fragment thereof.

In a further embodiment the kit comprises a reference compound is selected from the group consisting of astemizole, terfenadine, erythromycin, sparfloxain, probucol, terodiline and sertindole. In a preferred embodiment the reference compound is astemizole. It is a further object of this invention to provide kits wherein the reference compound is labeled, preferably radiolabeled.

In a specific embodiment the isolated and purified HERG polypeptide channel is bound to a solid support, preferably to a fluorescer cormprising solid support such as coated scintillation proximity beads.

Thus, in a specific embodiment the kit comprises a) an isolated and purified HERG polypeptide channel or a fragment thereof, bound to a solid support; and b) a labeled reference compound. Preferably this specific embodiment consists of a kit comprising a) an isolated and purified HERG polypeptide channel consisting of the amino acid sequence of SEQ ID NO:2, bound to fluorescer comprising solid support; and b) a radiolabeled reference compound, preferably [³H]-labeled astemizole.

In another specific embodiment the kit comprises a) membrane preparations of cells, preferably HEK293 cells, expressing on the surface thereof the HERG polypeptide channel consisting of the amino acid sequence of SEQ ID NO:2; b) [³H]-labeled astemizole; and c) means for measurement of the amount of labeled reference compound bound to HERG.

The means of measurement consist of separating means to remove the excess of unbound labeled reference compound from the incubation mixture and of means for detection of the labeled reference compound. The person skilled in the art will know the separating means available for removing the excess of unbound labeled reference compound from the incubation mixture. Said separating means include, but are not limited to, magnetic beads, centrifugation techniques and filtration techniques. The means for detecting the labeled reference compound will be dependend on the labeled used. Said labels may be fluorescent or radiolabels. The skilled man will know the detection means available depending on the label used.

In a specific embodiment the separating means consists of GF/B filtration (Whatman Inc, Clifton, N.J.). In another specific embodiment the detection means consists of scintillation counting in a Topcount (Packard, Meriden, Conn.).

In a further embodiment the kits of the invention further comprise instructions and/or multiple well plates for performing the assay.

This invention will be better understood by reference to the Experimental Details that follow, but those skilled in the art will readily appreciate that these are only illustrative of the invention as described more fully in the claims that follow thereafter. Additionally, throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.

EXAMPLE 1 DNA Constructs and Stable Transfection of HEK293 Cells

HERG cDNA (Genbank Accession number: U04270 (SEQ ID NO:1)) was subcloned into bamHI/EcoRI sites of the pcDNA3 vector (Invitrogen). This vector contains a CMV promotor and a SV40 promotor, which drive the expression of the inserted cDNA (HERG) and neomycin resistance gene, respectively. The HEK293 cells were transfected with this construct by a calcium phosphate precipitate method (Gibco) or a lipofectamine method (Gibco). After selection in 800 μg/ml geneticin (G418; Gibco) for 15-20 days, single colonies were picked with cloning cylinders and tested for HERG current. The stably transfected cells were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum and 400 μg/ml geneticin.

For electrophysiological study, the cells were harvested from the culture dish by trypsinization, washed twice with standard MEM medium and seeded on small petri-dishes coated with poly-L-lysine. Experiments were performed on the cells 1-2 days after plating.

EXAMPLE 2 Membrane Preparations of HEK293 Cells Stably Transfected with the HERG Potassium Channel

HEK293 cells stably transfected with the HERG channel cDNA, were grown in DMEM culture medium enriched with 10% fetal calf serum and antibiotics. Collected cells were homogenized in Tris-HCl 50 mM pH 7.4 using an Ultraturrax homogenizer and the homogenate was centrifuged for 10 min at 23,500×g in a Sorvall centrifuge. The cell membranes were washed once by re-homogenization and re-centrifugation. The membranes were supended inTris-HCl 50 mM pH 7.4, aliquoted and stored at −80° C.

EXAMPLE 3 Radiolabeling of Astemizole

A solution of 4.6 g of astemizole (I) (10 mmol) in a 48% aqueous hydrobromic acid solution (80 ml) was stirred and refluxed for 2 hours. The reaction mixture was allowed to cool to room temperature and the formed precipitate was filtered and washed with water. The solids were dissolved in a mixture of N,N-dimethylformamide (20 ml) and water (20 ml) and the mixture was made alkaline by introducing slowly and with stirring a concentrated aqueous ammoniumhydroxide solution. Then water (100 ml) was added and the mixture was stirred for 1 h. The precipitate was filtered off and dried to the air for 18 h to yield desmethylastemizole (II).

From this amount a fraction was taken and thoroughly.purified in portions via preparative HPLC on a Hypersyl ODS (5 μm) bonded phase stainless steel column (7.1 mm ID×300 mm) to yield astemizole free desmethylastemizole. Detection took place at 282 nm and elution was performed isocratically with acetonitrile-water-diisopropylamine (56:44:0.2, v/v) at a flow rate of 4.0 ml/min.

A fraction of the HPLC purified desmethylastemizole (II) (26.7 mg, 60 μmol) was dissolved in N,N-dimethylformamide (1.0 ml). To this solution IN aqueous sodium hydroxide solution (60 μl, 60 μmol) was added. The mixture was stirred for 25 minutes at room temperature and added dropwise to a precooled solution (−78° C.) of [3³H]-methyliodide (370 MBq) in toluene. The reaction mixture was vortexed and then left without cooling for 3 hours. The toluene was evaporated from the reaction mixture on a waterbath of 40° C. at aspirator pressure and the residue was purified in portions via preparative HPLC as described above. The product containing fractions were combined and depleted to 70 ml with methanol to give [³H]-astemizole (III) with a total radioactivity of 198 MBq and a specific activity of 3.14 TBq/mmol (85 Ci/mmol).

EXAMPLE 4 Radioligand Binding Assay

Membranes were thawed and re-homogenized in incubation buffer (Hepes 10 nM pH 7.4, 40 mM KCl, 20 mM KH₂PO₄, 5 mM MgCl₂, 0.5 mM KHCO₃, 10 mM glucose, 50 mM glutamate, 20 mM aspartate, 14 mM heptanoic acid, 1 mM EGTA, 0.1% BSA) and 20-100 μg protein was incubated with [³H]-astemizole for 60 min at 25° C. with or without competitor followed by rapid filtration over GF/B filter using a Filtermatel 96 harvester (Packard, Meriden, Conn.). Filters were rinsed extensively with ice-cold rinse-buffer (Tris-HCl 25 mM pH 7.4, 130 mM NaCl, 5.5 mM KCl, 5 mM glucose, 0.8 mM MgCl₂, 50 μM CaCl₂, 0.1% BSA). Filter bound radioactivity was determined by scintillation counting in a Topcount (Packard, Meriden, Conn.) and results were expressed as counts per minute (cpm).

Initially, various parameters including buffer, radioligand and compound to determined non-specific binding, were investigated in order to select the optimal conditions. In a saturation binding experiment, increasing concentrations of [³H]-astemizole were incubated with membranes, re-suspended in buffer. Non-specific binding was measured in the presence of 10 μM R66204 (FIG. 1).

The effect of BSA and/or cyclodextrine present in the incubation buffer, and of various ways of compound addition prior to the experiment, was investigated by comparing the binding affinities of 22 reference compounds to the electrophysiology data. Compounds were dissolved in DMSO and further diluted in the same solvent using a MultiprobeII pipetting station (Packard, Meriden, Conn.). The final DMSO concentration in all experiments was 1%. From this analysis it appears that compounds can be added directly from the DMSO stock solution. Attempts to increase the solubility of the compounds by addition of BSA and/or cyclodextrin did not improve the correlation significantly.

EXAMPLE 5 Whole-cell Voltage Clamp Technique (Patch Clamp)

Solutions: The bath solution contained (in mM) 150 NaCl, 4 KCl, 5 glucose, 10 HEPES, 1.8 CaCl₂ and 1 MgCl₂ (pH 7.4 with NaOH). The pipette solution contained (in mM) 120 KCl, 5 EGTA, 10 HEPES, 4 MgATP, 0.5 CaCl₂ and 2 MgCl₂ (pH 7.2 with KOH). Compounds were dissolved in DMSO to obtain a stock solution of 10⁻² M or 10⁻¹ M. Control (=bath solution+DMSO) and test solutions (=bath solution+DMSO+compound to be tested) contained 0.3%, 0.1% or 0.03% DMSO. Test and control solutions were applied to the cell under study using an Y-tube system, allowing to rapidly change solutions (less than 0.5 s) in the vicinity of the cell under study.

Electrophysiological measurements: A Petri dish containing attached HEK293 cells expressing HERG was fixed on the stage of a Patch Clamp Tower. An inverted microscope was used to observe the cells. The Petri dish was constantly perfused with the bath solution at room temperature.

Patch pipettes were pulled from borosilicate glass capillaries using a horizontal Flaming/Brown micropipette puller without further fire-polishing. The microelectrodes used had an input resistance between 1.5 and 3 MQ when filled with the pipette solution.

The membrane current of the cells was measured at distinct membrane potentials with the patch clamp technique by means of an EPC-9 patch clamp amplifier. Data were acquired and analysed using the programs Pulse and Pulsefit (HEKA), DataAccess (Bruxton) and Igor (Wavemetrics). The current signals were low-pass filtered and subsequently digitised. The liquid junction potential was electronically corrected, before establishing the seal. After disruption of the membrane, the cell capacitance and the series resistance were compensated using the circuit of the EPC-9 patch clamp amplifier.

The holding potential was −80 mV. The HERG current (K⁺-selective outward current) was determined as the maximal tail current at −40 mV after a 2 second depolarization to +60 mV. Pulse cycling rate was 15 s. Before each test pulse a short pulse (0.5 s) from the holding potential to −60 mV was given to determine leak current. After establishing whole-cell configuration a 5 minute equilibration period allowed for internal perfusion of the cell with the pipette solution. Thereafter test pulses were given for 5 minutes to quantify the HERG current under control conditions. While continuing the pulse protocol, perfusion was switched from control solution to drug-containing solution. The effect of the drug was measured after 5 minutes of drug application. One to three concentrations of the drug were tested per cell (applied cumulatively).

Parameter analysis of the measurements: The HERG current was determined as the maximal tail current at −40 mV after a 2 second depolarization to +60 mV, starting from a holding potential of −80 mV.

During the initial 5 minutes measured in the presence of the control solution, the amplitude of the HERG-mediated membrane K⁺ current gradually decreased with time (run-down). In order to quantify accurately the extent of block by the compounds, this continuous run-down of the K⁺ current has to be taken into account. Therefore the time course of the K⁺ current (measured at −40 mV) was fitted exponentially to the initial period of 5 minutes in control solution and extrapolated for the remainder of the experiment. These extrapolations give the estimated amplitude of the current if no drug would have been given. To determine the extent of block by the compounds, the ratio of the measured current was calculated by dividing each measured current amplitude by the value of the fitted current at the same point in time.

EXAMPLE 6 Pharmacological Evaluation of the Binding assay

For the pharmacological evaluation of the binding assay, 322 compounds were tested at 8 concentrations, for their ability to inhibit [³H]-astemizole binding to the HERG channel and pIC₅₀-values were calculated by non-linear regression analysis. If pIC₅0 values were available, the rank order (Spearman) of the potencies for binding and patch clamp was compared.

If in the patch clamp assay, compounds only have been tested at <4 concentrations, a score was assigned to both binding- and patch clamp data according to the following criteria:

-   -   score 1: pIC50<6 or % blockade<50% at 10⁻⁶ M or higher     -   score 2: pIC50 between 6-8 or % blockade<50% between 10⁻⁶ and         10⁻⁸ M     -   score 3: pIC50>8 or % blockade>50% at 10⁻⁸ M or lower

The rank order of potencies of 42 reference compounds to displace the [³H]-astemizole binding from the HERG channel, correlates well with the electrophysiological data for the functional blockade of the rapid activating delayed rectifier K⁺ current (rsp=0.87) (FIG. 2).

For 94% of the compounds tested, the binding data correlate with the patch clamp data. In 2% of the cases the binding assay scored higher than the patch clamp assay, for the remaining 4% it is the other way around, i.e. the patch clamp assay scores higher than the binding assay.

In view of this good correlation between binding data and electrophysiological data it may be concluded that the radioligand binding assay can be used as a primary screening tool for the prediction of potential cardiovascular side-effects. 

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 42. Radiolabeled astemizole of formula (III)


43. A process for preparing radiolabeled astemizole as in claim 42 comprising: a) demethylating astemizole of formula (I) using a suitable reagent such as 48% aqueous hydrobromic acid:

b) reacting the intermediate of formula (II) as set forth above with [³H]-methyliodide (CT₃I) optionally in a reaction inert solvent and in the presence of a base to obtain said radiolabeled astemizole:


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